Nicotine addiction is the leading source of preventable death worldwide. The bottleneck in rational design of novel therapeutics for nicotine addiction is high-resolution structural information for nicotinic acetylcholine receptors. Specifically, the alpha4beta2 subtype of the nicotinic receptor plays an essential role in nicotine addiction. Nicotine selectively upregulates this neurotransmitter receptor in systems ranging from clonal cell lines to smokers' brains, and mice lacking the alpha4beta2 receptor exhibit dramatically attenuated nicotine self-administration behavior. The only specific therapy for smoking cessation distinct from nicotine replacement is a partial agonist for the alpha4beta2 nicotinic receptor. Unfortunately, these therapies are only modestly effective in helping people quit smoking. The goal of this proposal is to resolve this pressing biomedical problem by determining the structure of the alpha4beta2 nicotinic receptor by X-ray crystallography. The alpha4beta2 nicotinic receptor is a particularly daunting target for crystallographic studies because it is: (1) a eukaryotic membrane protein; (2) an obligate heteropentameric protein; and (3) its subunit stoichiometry is variable, being formed from either two alpha and three beta subunits, or three alpha and two beta subunits. This proposal is organized into two phases. In the first, exploratory (R21) phase, the goals are to (1) identify alpha4 and beta2 subunit constructs that combine to express at high levels and form stable pentameric receptors, (2) purify a single stoichiometric species of receptor and (3) obtain preliminary crystals. In the second (R33) phase, the goals are to (1) obtain well-diffracting crystals of the alpha4beta2 receptor and (2) determine its structure in complex with different classes of ligands.